It is difficult to achieve high expression yields of factor IX in heterologous, particularly transgenic, systems. For example, while the basic approach to .beta.-lactoglobulin-driven transgenic expression of human factor IX in the milk of transgenic animals such as sheep (as disclosed in WO-A-8800239) does work, the yields obtained are low. There seem to have been two main reasons for this:
Failure to Express
The use of factor IX cDNAs has generally proved a problem in terms of getting reasonable levels of the appropriate fIX transcript. This problem was partially solved by the transgene rescue approach (described in WO-A-9211358, "Increased Expression by a Second Transferred Sequence in Transgenic Organisms"). In this prior publication, cointegration of .beta.-lactoglobulin (BLG) with the human factor IX-encoding construct FIXD led to the production of lines of mice expressing high levels of FIXD mRNA. The milk of these animals, however, contained very little fIX.
Aberrant Splicing
Closer inspection of the FIXD mRNA transcripts in the BLG+FIXD mice showed that they were approximately 450 bp shorter than predicted. It was surmised that these are deleted internally most probably by an aberrant splice of
the mRNA (Clark et al., Bio/Technology 10 1450-1454 (1992)).
Splicing of human factor IX mRNA in liver cells has been discussed in J. Biol. Chem. 270, 5276-5281 (1994) (Kurachi et al). Here it is indicated that the presence of splicing signal sequences results in increased expression of factor IX since spliceosome complexes act to protect precursor mRNAs from random degradation before being transported out of the nucleus.